Among the disorders, which the invention concerns, are those involving abnormal and excessive bleeding due to destruction of blood platelets ("platelets").
The disorders include post-transfusion purpura ("PTP") and post-transfusion platelet refractoriness ("PTPR"), which are suffered by some persons who receive blood or platelets from other persons by transfusion or the like.
The disorders also include one that is suffered by fetuses and newborns and is known as "neonatal alloimmune thrombocytopenic purpura" ("NATP"). This disorder can cause death of fetuses and serious birth defects or death of newborns. NATP is estimated to affect about 1 in 2000 newborns. In NATP, fetal platelets, which enter the mother's blood stream, induce production in the mother of antibodies against fetal platelets. These maternal antibodies then pass with the mother's blood into the fetus and mediate destruction of platelets in the fetus.
A mother, whose fetus or newborn suffers from NATP, is at increased risk of suffering PTP or PTPR.
When platelets from a first human (a "donor") are introduced into the blood system of a second human (a "recipient"), by transfusion, through the placenta (in the case of fetal blood entering the mother), or the like, the recipient may mount an immune response against the platelets from the donor. Such an immune response is referred to as an "alloimmune" response, because it involves antibodies reacting against antigens of the same species. The alloimmune response to platelets is due to an immune response of the recipient against "alloantigens" (antigens of the same species as that mounting the immune response) on platelets from the donor. These alloantigens are on membrane glycoproteins that occur in the membranes, which define the outer surfaces of platelets ("platelet membranes"). Each of these membrane glycoproteins typically has a cytoplasmic domain or segment, which is the part of the glycoprotein exposed to the cytoplasm inside the platelet; a transmembrane domain, which is the part of the glycoprotein embedded in the platelet membrane; and an extracellular domain or segment, which is the part of the glycoprotein exposed to the outside of the platelet. It is thought that alloantibodies, which are generated in an alloimmune response against platelet alloantigens, interact with the extracellular domains of the alloantigens.
The platelet alloantigens that a person has are determined by the person's genetics. A donor, because of his or her genetics, may have a platelet alloantigen, which a recipient, who receives blood or platelets from the donor, does not have, because of the recipient's genetics. In such a situation, the immune system of the recipient may recognize as "foreign," and raise an immune response against, the platelet alloantigen, which the donor has but the recipient does not.
Membrane glycoprotein alloantigens have been characterized for both human red blood cells and human platelets. It is noteworthy, however, that they also occur on other cell types, such as leucocytes and endothelial cells, where they may also occasion various disorders on account of alloimmune responses.
Recognized classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when blood recipients have been exposed to blood from donors.
A recent review of human platelet alloantigen systems is provided by Newman and Goldberger, Bailliere's Clin. Haematol. 4, 869-888 (1991).
Several "systems" of platelet alloantigens have been characterized which are diallelic. For each of these systems, there are two alloantigens, each of which is provided by one of two alleles of the gene which is associated with the system. Because a gene occurs twice in the normal human genome, a person can be homozygous for one or the other of the alloantigens or heterozygous for the alloantigens in the diallelic systems. The alloantigens occur on glycoprotein molecules which are heterodimers, i.e., they consist, in each case, of one each of two glycoprotein subunits that differ, typically significantly, in amino acid sequence and other characteristics. For each of these diallelic systems which has been characterized at the level of protein and gene sequences, it has been found that the difference in sequence which gives rise to the alloantigen occurs on one of the two subunits of the associated heterodimeric glycoprotein.
One diallelic system of human platelet alloantigens is the Br.sup.a /Br.sup.b diallelic system associated with GPIa/IIa, a membrane glycoprotein "integrin," which occurs on platelets and various cell types, such as leukocytes, fibroblasts and epithelial cells, and each molecule of which consists of one glycoprotein subunit designated "GPIa" and one glycoprotein subunit designated "GPIIa." Kiefel et al., Blood 733: 2219-2223 (1989); Santoso at al., Br. J. Haematol. 72: 191-98 (1989). GPIa/IIa, which is also known as "integrin .alpha..sub.2 .beta..sub.1 " or "VLA-2,", is the collagen receptor on platelets and other cell types (e.g., lung fibroblasts). In unstimulated platelets, GPIa/IIa mediates Mg.sup.2+ -dependent platelet adhesion to collagen. Staatz et al., J. Cell. Biol. 108:1917-24 (1989). Br alloantigens have also been found on activated T lymphocytes (Santoso et al., Hum. Immunol. 24:237-46 (1989)) and endothelial cells (Giltay et al., Br. J. Haematol. 75:557-600 (1990)), which is consistent with the tissue distribution known for integrin .alpha..sub.2 .beta..sub. 1.
The frequencies for the Br alleles are 0.1110 for Br.sup.a and 0.8890 for Br.sup.b in the caucasian population. Thus, in this population, 19.8% are heterozygous for the alleles, and will not mount an alloimmune response due to Br incompatibility (not possessing a Br alloantigen on platelets received from another); 1.2% are homozygous for the Br.sup.a allele and may mount an immune response due to Br alloantigen incompatibility against platelets received from anyone in the 98.8% of the caucasian population that is not homozygous for the Br.sup.a allele; and 79.0% are homozygous for the Br.sup.b allele and may mount an immune response due to Br alloantigen incompatibility against platelets received from anyone in the 21.0% of the caucasian population that is not homozygous for the Br.sup.b allele.
As indicated above, alloimmunization based on Br incompatibility (i.e., introducing into the blood stream of a recipient platelets with a Br alloantigen which the recipient does not have) can result in bleeding disorders due to platelet destruction, such as NATP (Kiefel et al., Vox Sang. 54:101-06 (1987); Kiefel et al., Vox Sang. 60:244-45 (1991); Bettaieb et al., Vox Sang. 60:230-34 (1991), PTP (Christie et al., Blood 77:2785-89 (1991) and PTPR (Bierling et al., Br. J. Haematol. 73:428-29 (1989)). The location, in the GPIa/IIa molecule, or the nature of the polymorphism which underlies the Br.sup.a /Br.sup.b alloantigen system, and the genetic polymorphism, which underlies the polymorphism at the glycoprotein level, have not heretofore been known, although there has been speculation (Newman and Goldberger, supra) that the polymorphism resides on the GPIa subunit alone.
Furthermore, heretofore it has not been possible to obtain non-human antibody (polyclonal or monoclonal), as from a rat, mouse, goat, chicken, or the like, with specificity for the Br.sup.a alloantigen but not the Br.sup.b alloantigen or vice-versa sufficient for use of the antibody, in an immunoassay, for typing for Br phenotype using platelets or GPIa/IIa molecules.
The complete amino acid sequence of the GPIa portion of a GPIa/IIa complex has been deduced from the nucleotide sequence of the corresponding cDNA obtained from an human lung fibroblast cDNA library. Takada and Hemler, J. Cell Biol. 109: 397-407 (1989). The mature GPIa polypeptide has 1152 amino acids with, in lung fibroblasts, an extracellular domain that is said to have 1103 amino acids, a transmembrane domain, and a short cytoplasmic domain that is said to have 22 amino acids. Although the overall sequence homology between GPIa (an integrin .alpha..sup.2 subunit) and other integrin .alpha. subunits is only 18-25%, GPIa has a similar distribution of cysteine residues and cation-binding domains with these other subunits. GPIa also contains a 191 amino acid insert, called the "I-domain," which contains potential sites for interaction with collagen.
Previously developed technology, involving target nucleic acid amplification starting with platelet RNA and sequencing of the resulting amplified nucleic acid, has been applied with the currently recognized diallelic platelet alloantigen systems, other than the Br.sup.a /Br.sup.b system, to determine, at the nucleotide sequence level, the base differences and, at the amino acid sequence level, the amino acid differences between the two alleles of each system. Newman and Aster, U.S. Pat. No. 5,091,302; Newman et al., J. Clin. Invest. 82,739-744 (1988); Newman et al., J. Clin. Invest. 83, 1778-1781 (1989)(P1.sup.A system); Lyman et al., Blood 75, 2343-2348 (1990) (Bak system); Kuipers et al., J. Clin. Invest. 89, 381-384 (1992)(Ko system); Wang et al., J. Clin. Invest. 90, 2038-2043 (1992)(Pen system). In each of the Pl.sup.A, Bak, Ko and Pen systems, it has been found that an amino acid difference at a single position differentiates the subunits, that differ in amino acid sequence, in the two alleles and that a nucleotide difference at a single position differentiates the coding regions of the mRNAs (and presumably the genes) that encode these subunits.